Stimulation of Src family protein-tyrosine kinases as a proximal and mandatory step for SYK kinase-dependent phospholipase Cgamma2 activation in lymphoma B cells exposed to low energy electromagnetic fields.
Here, we present evidence that exposure of DT40 lymphoma B cells to low energy electromagnetic field (EMF) results in a tyrosine kinase-dependent activation of phospholipase Cgamma2 (PLC-gamma2) leading to increased inositol phospholipid turnover. B cells rendered PLC-gamma2-deficient by targeted disruption of the PLC-gamma2 gene as well as PLC-gamma2-deficient cells reconstituted with Src homology domain 2 (SH2) domain mutant PLC-gamma2 did not show any increase in inositol-1,4,5-trisphosphate levels after EMF exposure, providing direct evidence that PLC-gamma2 is responsible for EMF-induced stimulation of inositol phospholipid turnover, and its SH2 domains are essential for this function. B cells rendered SYK-deficient by targeted disruption of the syk gene did not show PLC-gamma2 activation in response to EMF exposure. The C-terminal SH2 domain of SYK kinase is essential for its ability to activate PLC-gamma2. SYK-deficient cells reconstituted with a C-terminal SH2 domain mutant syk gene failed to elicit increased inositol phospholipid turnover after EMF exposure, whereas SYK-deficient cells reconstituted with an N-terminal SH2 domain mutant syk gene showed a normal EMF response. LYN kinase is essential for the initiation of this biochemical signaling cascade. Lymphoma B cells rendered LYN-deficient through targeted disruption of the lyn gene did not elicit enhanced inositol phospholipid turnover after EMF exposure. Introduction of the wild-type (but not a kinase domain mutant) mouse fyn gene into LYN-deficient B cells restored their EMF responsiveness. B cells reconstituted with a SH2 domain mutant fyn gene showed a normal EMF response, whereas no increase in inositol phospholipid turnover in response to EMF was noticed in LYN-deficient cells reconstituted with a SH3 domain mutant fyn gene. Taken together, these results indicate that EMF-induced PLC-gamma2 activation is mediated by LYN-regulated stimulation of SYK, which acts downstream of LYN kinase and upstream of PLC-gamma2.
Dibirdik
I
I
Biotherapy Program, University of Minnesota, Minneapolis, Minnesota 55417, USA.
Kristupaitis
D
D
Kurosaki
T
T
Tuel-Ahlgren
L
L
Chu
A
A
Pond
D
D
Tuong
D
D
Luben
R
R
Uckun
F M
FM
D000818:Animals; D001402:B-Lymphocytes / Q000201:enzymology*; D002645:Chickens; D004574:Electromagnetic Fields; D004789:Enzyme Activation / Q000502:physiology; D004792:Enzyme Precursors / Q000172:deficiency / Q000235:genetics*; D018390:Gene Targeting; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes / Q000378:metabolism*; D008223:Lymphoma; D010716:Phosphatidylinositols / Q000378:metabolism; D051966:Phospholipase C gamma; D011505:Protein-Tyrosine Kinases / Q000172:deficiency / Q000235:genetics*; D011518:Proto-Oncogene Proteins / Q000032:analysis / Q000235:genetics; D051076:Proto-Oncogene Proteins c-fyn; D015398:Signal Transduction / Q000502:physiology; D000072377:Syk Kinase; D010738:Type C Phospholipases / Q000378:metabolism*; D018909:src Homology Domains / Q000502:physiology; D019061:src-Family Kinases / Q000378:metabolism*
D004792:Enzyme Precursors; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; D010716:Phosphatidylinositols; D011518:Proto-Oncogene Proteins; D015544:Inositol 1,4,5-Trisphosphate; D011505:Protein-Tyrosine Kinases; D051076:Proto-Oncogene Proteins c-fyn; D000072377:Syk Kinase; C000606897:Syk protein, mouse; D019061:src-Family Kinases; D010738:Type C Phospholipases; D051966:Phospholipase C gamma